Divalent-metal selectivity of Nramp1HA and Nramp2HA. The divalent-metal selectivity of Nramp1HA and Nramp2HA was compared in dose-response experiments. Transfected CHO cell lines N1-94 (Nramp1HA) and N2-310a (Nramp2HA), along with untransfected CHO cells, were resuspended in transport buffer (3 × 10⁶ cells/0.375 mL), and transport was initiated by addition of an equal volume of radioisotope buffer containing tracer amounts of ⁵⁵Fe²⁺ (A) or ⁵⁴Mn²⁺ (B), followed by incubation at 20°C. The final concentration of Fe²⁺ and Mn²⁺ in the transport reaction was varied between 0.31 μM and 10 μM total divalent metal (by 2-fold serial dilution), while the specific radioactivity of each was held constant. For Fe²⁺ transport buffer, a 50:1 molar excess of ascorbate to Fe²⁺ was used and kept constant at each Fe²⁺ concentration. Transport was allowed to proceed for 10 minutes at 20°C, and cell-associated radioactivity was determined as described in “Materials and methods” and in the legend to Figure 6. Parallel transport assays were conducted at 4°C, and these values were subtracted from those obtained at 20°C to determine the net amount of metal uptake, which is expressed as pmoleq of divalent-metal/μg of total cellular protein.